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Aim: Insulin therapy require self-administration of subcutaneous injection leading to painful and inconvenient drug therapy. The aim is to fabricate nanoemulsion (NE) based insulin loaded microneedles with improved bioavailability and patient compliance. Materials & methods: Different ratios of polyvinyl alcohol and polyvinylpyrrolidone as polymers were prepared through micro-molding technique for microneedles. Characterization of were performed using scanning electron microscope, differential scanning calorimetry, Fourier-transform infrared spectroscopy and circular dichroism. Mechanical strength, hygroscopicity and pain perception of these microneedles were also evaluated. In vitro release, permeation and in vivo PK/PD study of NE-based microneedles were conducted. Results: NE-based microneedles of insulin have improved bioavailability and quick response. Conclusion: Microneedles loaded with insulin can be effectively delivered insulin transdermally to treat diabetes with increased convenience and patient compliance.
[Box: see text].
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Organic-inorganic hybrid nanomaterials are considered as promising immobilization matrix for enzymes owing to their markedly enhanced stability and reusability. Herein, collagenase was chosen as a model enzyme to synthesize collagenase hybrid nanoflowers (Col-hNFs). Maximum collagenase activity (155.58 µmol min-1 L-1) and encapsulation yield (90 %) were observed in presence of Zn(II) ions at 0.05 mg/mL collagenase, 120 mM zinc chloride and PBS (pH 7.5). Synthesized Col-Zn-hNFs were extensively characterized by scanning electron microscopy (SEM), energy dispersive X-ray (EDX), X-ray diffraction (XRD), Fourier transform infrared (FTIR), circular dichroism (CD), fluorescence spectroscopy, dynamic light scattering (DLS) and zeta potential measurements. SEM images showed flower-like morphology with average size of 5.1 µm and zeta potential of -14.3 mV. Col-Zn-hNFs demonstrated superior relative activity across wide pH and temperature ranges, presence of organic solvents and surfactants as compared to its free form. Moreover, Col-Zn-hNFs exhibited excellent shelf life stability and favorable reusability. Col-Zn-hNFs showed the ability to suppress and eradicate fully developed insulin fibrils in vitro (IC50 = 2.8 and 6.2 µg/mL, respectively). This indicates a promising inhibitory potential of Col-Zn-hNFs against insulin amyloid fibrillation. The findings suggest that the utilization of Col-Zn-hNFs as a carrier matrix holds immense potential for immobilizing collagenase with improved catalytic properties and biomedical applications.
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Colagenases , Estabilidade Enzimática , Enzimas Imobilizadas , Nanoestruturas , Proteólise , Colagenases/metabolismo , Colagenases/química , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Nanoestruturas/química , Concentração de Íons de Hidrogênio , Amiloide/química , Temperatura , Insulina/química , Zinco/químicaRESUMO
Background and Aim: Intubation with Macintosh requires flexing the lower cervical spine and extending the atlanto-occipital joint to create a "line of sight." Primary aim of study was to compare the extent of cervical spine movement during laryngoscopy using conventional Macintosh laryngoscope and Airtraq. Material and Methods: A total of 25 patients of either sex between the age group of 18 and 60 years, having American Society of Anesthesiologists (ASA) physical status of Grade-I and Grade-II, scheduled for elective surgery under image control requiring general anesthesia and intubation were enrolled. A baseline image of the lateral cervical spine including the first four cervical vertebrae was taken by an image intensifier. After administration of general anesthesia, laryngoscopy was first performed using a Macintosh laryngoscope and a second X-ray image of the lateral cervical spine was taken. The second laryngoscopy using a Airtraq laryngoscope was done and the third image of the lateral cervical spine was taken. Angles between occiput and C1; C1 and C2; C2 and C3; C3 and C4; and occiput and C4 were calculated. Atlanto-occipital distance (AOD) was calculated as the distance between occiput and C1. Results: Macintosh showed greater cervical movement as compared with Airtraq but a significant difference in the movement was observed at C2-C3 and C0-C4. Baseline mean AOD was 2.21 ± 1.25 mm, after Macintosh and Airtraq laryngoscopy was found to be 1.13 ± 0.60 and 1.6 ± 0.78 mm, respectively, and was found to be significant (P < 0.05). Conclusion: We conclude that Airtraq allows intubation with less movement of the upper cervical spine makes Airtraq preferred equipment for intubation in patients with a potential cervical spine injury.
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Background: International students are often exposed to various challenges during life transitions. The 'mindsponge' mechanism suggests that individuals absorb and integrate new cultural values that are compatible with their core values while rejecting those of lesser importance. On the basis of this notion, this article explores the experiences of international students in China regarding their unplanned return to their home countries during the COVID-19 pandemic through the lens of the mindsponge mechanism. Aim: This article aims to highlight the experiences of international students in China who are going through life transitions due to the global pandemic. The study focuses on the experiences of two groups of international students: (1) Those who remained in China during the pandemic, and (2) those who had left China and were stranded in their home countries due to a ban on international travel amid COVID-19. Method: This qualitative study comprised of in-depth semi structured in-person and online interviews. Thematic Analysis was used to analyze the data in order to generate study themes. Results: The results revealed that students who remained in China experienced challenges which included anxiety, closure of campuses, lockdown, their parents' concern regarding health issues, and not being able to meet with friends. On the other hand, students who had left China during the pandemic were confined to their home countries. This group of students experienced more severe problems than the students who remained in China. Since the transition to home countries was "unplanned," they were not ready to readjust to their native culture and were vulnerable to severe reverse culture shock. Upon returning to their home countries, international students faced a number of challenges, including readjustment to their home countries and changes in their lives in host and home countries. In addition, they lost social and academic resources, such as the disruption of study environment, losing important group memberships, financial constraints, visa expiry, graduation delay, and academic suspension. Conclusion: This study concluded that the international students experienced cultural problems after unplanned transition to their home countries during the pandemic. They described effects of reverse culture shock as being more distressing. They perceived dissatisfaction due to loss of previously held social identities and sense of belonging to the traditional society they left behind. There is a need of future studies on the long-term effect of unplanned transition on psychological, social and professional experiences. The process of readjustment has proven to be a challenging endeavor.
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HIV-1 encodes accessory proteins that neutralize antiviral restriction factors to ensure its successful replication. One accessory protein, the HIV-1 viral infectivity factor (Vif), is known to promote ubiquitination and proteasomal degradation of the antiviral restriction factor apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (APOBEC3G), a cytosine deaminase that leads to hypermutations in the viral DNA and subsequent aberrant viral replication. We have previously demonstrated that the HIV-1 viral transcription mediator Tat activates the host progrowth PI-3-AKT pathway, which in turn promotes HIV-1 replication. Because the HIV-1 Vif protein contains the putative AKT phosphorylation motif RMRINT, here we investigated whether AKT directly phosphorylates HIV-1 Vif to regulate its function. Coimmunoprecipitation experiments showed that AKT and Vif interact with each other, supporting this hypothesis. Using in vitro kinase assays, we further showed that AKT phosphorylates Vif at threonine 20, which promotes its stability, as Vif becomes destabilized after this residue is mutated to alanine. Moreover, expression of dominant-negative kinase-deficient AKT as well as treatment with a chemical inhibitor of AKT increased K48-ubiquitination and proteasomal degradation of HIV-1 Vif. In contrast, constitutively active AKT (Myr-AKT) reduced K48-ubiquitination of Vif to promote its stability. Finally, inhibition of AKT function restored APOBEC3G levels, which subsequently reduced HIV-1 infectivity. Thus, our results establish a novel mechanism of HIV-1 Vif stabilization through AKT-mediated phosphorylation at threonine 20, which reduces APOBEC3G levels and potentiates HIV-1 infectivity.
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Desaminase APOBEC-3G , Infecções por HIV , HIV-1 , Produtos do Gene vif do Vírus da Imunodeficiência Humana , Desaminase APOBEC-3G/genética , Desaminase APOBEC-3G/metabolismo , Infecções por HIV/fisiopatologia , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Humanos , Fosforilação , Estabilidade Proteica , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Treonina/metabolismo , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Background and Aims: Video laryngoscopy has been shown to improvise Cormack-Lehane grading and rate of successful tracheal intubation and is now incorporated in most of the difficult airway guidelines. Since there is scarce literature regarding the use of channeled blade of King Vision video laryngoscope (KVVL) in anticipated difficult intubation, we planned to undertake this randomized control trial to assess the performance of channeled blade of KVVL and Macintosh laryngoscope in patients with anticipated difficult intubation. Design and Setting: This prospective randomized study was conducted in a tertiary care hospital. Materials and Methods: Patients fulfilling the inclusion criteria were randomly assigned equally to the KVVL group or Macintosh group. The primary outcome of the study was intubation success in the first attempt and number of attempts required for intubation, and the secondary outcomes were Cormack-Lehane grading and time required to intubate in both the groups. Results: The first-pass success of intubation was 88.6% in the KVVL group and 76.5% in the Macintosh group (P = 0.035). The second attempt of intubation was required in 11.4% and 20.6% of patients in the KVVL and Macintosh groups, respectively. Cormack Lehane Grade I was achieved in 100% of patients of the KVVL group as compared to 29.4% of patients in the Macintosh group. Moreover, the difference was statistically significant (P = 0.035). The mean duration of intubation was prolonged in the KVVL group as compared to the Macintosh group, and the difference was statistically significant (P = 0.04). Conclusion: The channeled blade of KVVL had a higher first-pass success rate and required fewer attempts to intubate when used in patients with anticipated difficult intubation. Further, the KVVL was found to be significantly better than the Macintosh laryngoscope in terms of Cormack-Lehane grading, but the time taken to intubate the trachea was more in the KVVL group.
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Mammalian innate immune response to virus infection is meditated by many cell-intrinsic pathways. RNA viruses, such as Sendai virus, which replicate in the cytoplasm, trigger the RIG-I-like receptor pathway, which activates the transcription factor, IRF3. Activated IRF3 translocates to the nucleus and induces transcription of the genes which encode interferons, the major antiviral cytokines. Interestingly, IRF3 activates another interferon-independent antiviral pathway, called RIG-I induced pathway of apoptosis (RIPA). For activating RIPA, IRF3 translocates from the cytoplasm to mitochondria. RIPA requires linear polyubiquitination of IRF3 by the enzyme complex, LUBAC; ubiquitinated IRF3 binds to Bax and translocates it to mitochondria causing the release of Cytochrome C, activation of caspases and apoptosis of the infected cell. Here, we report that Otulin, the deubiquitinase that removes linear polyubiquitin chains, inhibits RIPA by deubiquitinating IRF3. Ablation of Otulin expression enhanced RIPA and its overexpression inhibited RIPA. In virus-infected cells, to overcome Otulin-mediated inhibition, RIPA actively degrades Otulin. This degradation required sequential actions of RIPA-activated Caspase 3 and proteasomes. Caspase 3 cleaved Otulin at D31; the D31A mutant was not cleaved at all. The caspase-cleaved fragment was totally degraded by proteasomes, which was preceded by its K48-linked ubiquitination. Mass spectrometric analysis of Otulin identified K64 and K197 as the ubiquitinated residues. Otulin interacted with LUBAC after virus infection and the E3-ubiquitin ligase, HOIP, a component of LUBAC, ubiquitinated Otulin to trigger its proteasome-mediated degradation. To assess the impact of Otulin degradation on RIPA-mediated antiviral action, we expressed, in Otulin-ablated cells, a non-degradable mutant of Otulin, in which D31, K64 and K197 had been mutated. The cells expressing the Otulin mutant were less susceptible to virus-induced apoptosis, because RIPA was less active, and consequently virus replication was more robust. Thus, our study has revealed an important positive feedback loop of RIPA.
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Antivirais , Viroses , Animais , Apoptose , Caspase 3/metabolismo , Mamíferos/metabolismo , Ubiquitina/metabolismo , UbiquitinaçãoRESUMO
HCV core protein is the first structural protein synthesized during hepatitis C virus (HCV) infection and replication. It is released from virus infected liver cells and mediates multiple functions to affect host cell response. The innate immune response is the first line of defense against viral infection. After HCV infection, Kupffer cells (KCs) which are liver macrophages play an important role in host innate immune response. Kupffer cells act as phagocytes and release different cytokines and chemokines to counter viral infection and regulate inflammation and fibrosis in liver. Earlier, we have demonstrated that HCV core protein interacts with gC1qR and activates MAPK, NF-κB and PI3K/AKT pathways in macrophages. In this study, we explored the effect of HCV core protein on CCL2 and CXCL10 expression in macrophages and the signaling pathways involved. Upon silencing of gC1qR, we observed a significant decrease expression of CCL2 and CXCL10 in macrophages in the presence of HCV core protein. Inhibiting NF-κB pathway, but not P38, JNK, ERK and AKT pathways greatly reduced the expression of CCL2 and CXCL10. Therefore, our results indicate that interaction of HCV core protein with gC1qR could induce CCL2 and CXCL10 secretion in macrophages via NF-κB signaling pathway. These findings may shed light on the understanding of how leukocytes migrate into the liver and exaggerate host-derived immune responses and may provide novel therapeutic targets in HCV chronic inflammation.
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Quimiocina CCL2/imunologia , Quimiocina CXCL10/imunologia , Hepacivirus/imunologia , Macrófagos/imunologia , NF-kappa B/imunologia , Transdução de Sinais/imunologia , Proteínas do Core Viral/imunologia , Animais , Células Cultivadas , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Quimiocina CXCL10/genética , Quimiocina CXCL10/metabolismo , Expressão Gênica/imunologia , Hepacivirus/metabolismo , Hepacivirus/fisiologia , Hepatite C/imunologia , Hepatite C/metabolismo , Hepatite C/virologia , Interações Hospedeiro-Patógeno/imunologia , Humanos , Células de Kupffer/imunologia , Células de Kupffer/metabolismo , Células de Kupffer/virologia , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos BALB C , Células RAW 264.7 , Células THP-1 , Proteínas do Core Viral/metabolismoRESUMO
The innate immune response is crucial for defense against viral infections. Cells recognize virus infection through pattern recognition receptors and induce type I interferons as well as proinflammatory cytokines to orchestrate an innate immune response. Herpes simplex virus 1 (HSV-1) triggers both the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) and Toll-like receptor 3 (TLR3) pathways. It is well known that TLR3 uses the adaptor protein Toll/interleukin-1 receptor (IL-1R) domain-containing adaptor-inducing beta interferon (TRIF) for signaling, but we recently reported that STING signaling also requires TRIF. Because STING directly binds to TRIF, we identified the STING-interacting domain of TRIF and generated STING-noninteracting mutants of human and mouse TRIFs. The mutant TRIFs were unable to support STING signaling, although they were fully functional in the TLR3 pathway. These mutants were used to assess the relative contributions of the TLR3 and STING pathways to the attenuation of HSV-1 replication in mouse and human cell lines. For this purpose, the mouse L929 and NB41A3 cell lines and the human HT1080 and HeLa-M cell lines, in which both the TLR3 and the STING pathways are operational, were used. The TRIF gene was disrupted in these lines by CRISPR/Cas9, before reconstituting them with mutant and wild-type TRIF expression vectors. Infection of the reconstituted cells with HSV-1 revealed that both the cGAS-STING and the TLR3 signaling pathways are required for the attenuation of virus replication, but their relative contributions in attenuating HSV-1 replication were found to be different in mouse versus human cell lines. Thus, our study suggests that the relative contributions of the cGAS-STING and the TLR3 pathways in the attenuation of viral infection may be species specific.IMPORTANCE The magnitude of fatal infections caused by all different viruses in human and animal populations justifies a better understanding of the host innate immune response process that attenuates virus replication. In particular, the relative contributions of different signaling pathways which are responsible for the generation of the innate immune response are still largely unknown. In this study, we used STING-noninteracting TRIF mutants to decipher the relative contributions of the TLR3 and cGAS-STING signaling pathways to the attenuation of HSV-1 infection. We show that the relative contributions of the two pathways to the attenuation of viral infection are different in mouse versus human cell lines. Together, our results provide new insights into the relative contributions of two different signaling pathways in the attenuation of viral infection and may lead to the development of new antiviral strategies aimed at blocking viral infection at very early stages.
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Herpesvirus Humano 1/fisiologia , Proteínas de Membrana/metabolismo , Nucleotidiltransferases/metabolismo , Transdução de Sinais , Receptor 3 Toll-Like/metabolismo , Replicação Viral , Animais , Células HEK293 , Células HeLa , Humanos , Proteínas de Membrana/genética , Camundongos , Receptor 3 Toll-Like/genéticaRESUMO
OBJECTIVES: The diagnosis and management of oral cavity cancers are often complicated by the uncertainty of which patients will undergo malignant transformation, obligating close surveillance over time. However, serial biopsies are undesirable, highly invasive, and subject to inherent issues with poor inter-pathologist agreement and unpredictability as a surrogate for malignant transformation and clinical outcomes. The goal of this study was to develop and evaluate a Multivariate Analytical Risk Index for Oral Cancer (MARIO) with potential to provide non-invasive, sensitive, and quantitative risk assessments for monitoring lesion progression. MATERIALS AND METHODS: A series of predictive models were developed and validated using previously recorded single-cell data from oral cytology samples resulting in a "continuous risk score". Model development consisted of: (1) training base classification models for each diagnostic class pair, (2) pairwise coupling to obtain diagnostic class probabilities, and (3) a weighted aggregation resulting in a continuous MARIO. RESULTS AND CONCLUSIONS: Diagnostic accuracy based on optimized cut-points for the test dataset ranged from 76.0% for Benign, to 82.4% for Dysplastic, 89.6% for Malignant, and 97.6% for Normal controls for an overall MARIO accuracy of 72.8%. Furthermore, a strong positive relationship with diagnostic severity was demonstrated (Pearson's coefficientâ¯=â¯0.805 for test dataset) as well as the ability of the MARIO to respond to subtle changes in cell composition. The development of a continuous MARIO for PMOL is presented, resulting in a sensitive, accurate, and non-invasive method with potential for enabling monitoring disease progression, recurrence, and the need for therapeutic intervention of these lesions.
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Citodiagnóstico , Neoplasias Bucais/diagnóstico , Biópsia , Citodiagnóstico/instrumentação , Citodiagnóstico/métodos , Citodiagnóstico/normas , Humanos , Dispositivos Lab-On-A-Chip , Análise Multivariada , Gradação de Tumores , Estadiamento de Neoplasias , Reprodutibilidade dos Testes , Medição de RiscoRESUMO
Despite the high success rate, antiretroviral therapy does not cure the disease completely due to presence of latent viral reservoirs. Although several studies have addressed this issue earlier, the role of serum starvation/deprivation in HIV-1 latency has not been studied. So, we investigated the role of serum starvation in regulating HIV-1 latency. The impact of serum starvation on HIV-1 latency was assessed in latently infected monocytes U1 and T-cells J1.1. Serum starvation breaks HIV-1 latency in U1 cells. Under similar conditions, J1.1 cells failed to show reactivation of virus. We investigated the involvement of cell death pathway and autophagy during the serum starvation in viral reactivation. Inhibition of these pathways did not affect viral reactivation. Furthermore, other crucial factors like NF-κB, SP1 and AKT did not play any role in regulating viral latency. Here, we report that serum deprivation up-regulates ERK/JNK pathway. This leads to phosphorylation of c-Jun which plays an important role in viral reactivation. Treatment of cells with U0126, an ERK kinase inhibitor, potently inhibited viral replication. In summary, we show that serum starvation leads to reactivation of HIV-1 in latently infected monocytes through the ERK/JNK pathway.
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Infecções por HIV/enzimologia , HIV-1/fisiologia , Sistema de Sinalização das MAP Quinases , Monócitos , Ativação Viral/fisiologia , Latência Viral/fisiologia , Autofagia , Linhagem Celular , Infecções por HIV/patologia , Humanos , Monócitos/enzimologia , Monócitos/patologia , Monócitos/virologia , Ativação Viral/efeitos dos fármacos , Latência Viral/efeitos dos fármacosRESUMO
Fanconi anemia (FA) is a hereditary genomic instability disorder with a predisposition to leukemia and oral squamous cell carcinomas (OSCCs). Hematopoietic stem cell transplantation (HSCT) facilitates cure of bone marrow failure and leukemia and thus extends life expectancy in FA patients; however, survival of hematologic malignancies increases the risk of OSCC in these patients. We developed a "cytology-on-a-chip" (COC)-based brush biopsy assay for monitoring patients with oral potentially malignant disorders (OPMDs). Using this COC assay, we measured and correlated the cellular morphometry and Minichromosome Maintenance Complex Component 2 (MCM2) expression levels in brush biopsy samples of FA patients' OPMD with clinical risk indicators such as loss of autofluorescence (LOF), HSCT status, and mutational profiles identified by next-generation sequencing. Statistically significant differences were found in several cytology measurements based on high-risk indicators such as LOF-positive and HSCT-positive status, including greater variation in cell area and chromatin distribution, higher MCM2 expression levels, and greater numbers of white blood cells and cells with enlarged nuclei. Higher OPMD risk scores were associated with differences in the frequency of nuclear aberrations and differed based on LOF and HSCT statuses. We identified mutation of FAT1 gene in five and NOTCH-2 and TP53 genes in two cases of FA patients' OPMD. The high-risk OPMD of a non-FA patient harbored FAT1, CASP8, and TP63 mutations. Use of COC assay in combination with visualization of LOF holds promise for the early diagnosis of high-risk OPMD. These minimally invasive diagnostic tools are valuable for long-term surveillance of OSCC in FA patients and avoidance of unwarranted scalpel biopsies.
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INTRODUCTION: Congenital variants of the cervical spine may mimic traumatic lesions and may cause recurrent episodes of pain. The spectrum of cervical variants includes persistent apophyses of the transverse processes, persistent epiphyses, vertebral platyspondylia, vertebral hypoplasia, and dysplasia of the vertebral arch. Furthermore, abnormalities of the spinous process have been described including doubled spinous processes and hypertrophies. Unilateral hyperplasia of a spinous process is a rare finding that has only been described rarely as case reports. CASE REPORT: We report a 9-year-old male child who was referred to us with swelling in the posterior aspect of the neck. Anteroposterior and lateral radiographs of the cervical spine show an elongated left spinous process in the neck at the level of C5 vertebrae. There was an associated hemivertebra at the C4 level. Computed tomography examination better depicted this congenital variant and clearly showed the associated schisis of the posterior arch as well as unfused spinous process at the same level on the left side. This is a very rare congenital anomaly and probably among the few such cases reported in literature. CONCLUSION: Rare congenital spinal abnormalities including unilateral hyperplasia of a spinous process have to be kept in mind as a differential diagnosis in patients with posterior midline neck swelling and recurrent episodes of cervical neck pain.
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Murine double minute 2 (Mdm2) is known to enhance the transactivation potential of human immunodeficiency virus (HIV-1) Tat protein by causing its ubiquitination. However, the regulation of Mdm2 during HIV-1 infection and its implications for viral replication have not been well studied. Here, we show that the Mdm2 protein level increases during HIV-1 infection and this effect is mediated by HIV-1 Tat protein. Tat appears to stabilise Mdm2 at the post-translational level by inducing its phosphorylation at serine-166 position through AKT. Although p53 is one of the key players for Mdm2 induction, Tat-mediated stabilisation of Mdm2 appears to be independent of p53. Moreover, the non-phosphorylatable mutant of Mdm2 (S166A) fails to interact with Tat and shows decreased half-life in the presence of Tat compared with wild-type Mdm2. Furthermore, the non-phosphorylatable mutant of Mdm2 (S166A) is unable to support HIV-1 replication. Thus, HIV-1 Tat appears to stabilise Mdm2, which in turn enhances Tat-mediated viral replication. This study highlights the importance of post-translational modifications of host cellular factors in HIV-1 replication and pathogenesis.
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HIV-1/fisiologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Mutação , Fosforilação , Proteínas Proto-Oncogênicas c-mdm2/genética , RNA Interferente Pequeno/genética , Proteína Supressora de Tumor p53/metabolismo , Replicação ViralRESUMO
Deubiquitinases (DUBs) are key regulators of complex cellular processes. HIV-1 Tat is synthesized early after infection and is mainly responsible for enhancing viral production. Here, we report that one of the DUBs, USP7, stabilized the HIV-1 Tat protein through its deubiquitination. Treatment with either a general DUB inhibitor (PR-619) or USP7-specific inhibitor (P5091) resulted in Tat protein degradation. The USP7-specific inhibitor reduced virus production in a latently infected T-lymphocytic cell line J1.1, which produces large amounts of HIV-1 upon stimulation. A potent increase in Tat-mediated HIV-1 production was observed with USP7 in a dose-dependent manner. As expected, deletion of the USP7 gene using the CRISPR-Cas9 method reduced the Tat protein and supported less virus production. Interestingly, the levels of endogenous USP7 increased after HIV-1 infection in human T-cells (MOLT-3) and in mammalian cells transfected with HIV-1 proviral DNA. Thus, HIV-1 Tat is stabilized by the host cell deubiquitinase USP7, leading to enhanced viral production, and HIV-1 in turn up-regulates the USP7 protein level.
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HIV-1/fisiologia , Linfócitos T/metabolismo , Ubiquitina Tiolesterase/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Aminopiridinas/farmacologia , Fármacos Anti-HIV/farmacologia , Sistemas CRISPR-Cas , Linhagem Celular , Indução Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Genes Reporter/efeitos dos fármacos , Células HEK293 , HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Células Jurkat , Estabilidade Proteica/efeitos dos fármacos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Linfócitos T/efeitos dos fármacos , Linfócitos T/imunologia , Linfócitos T/virologia , Tiocianatos/farmacologia , Tiofenos/farmacologia , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/genética , Peptidase 7 Específica de Ubiquitina , Ubiquitinação/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/química , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
UNLABELLED: Despite significant advances in surgical procedures and treatment, long-term prognosis for patients with oral cancer remains poor, with survival rates among the lowest of major cancers. Better methods are desperately needed to identify potential malignancies early when treatments are more effective. OBJECTIVE: To develop robust classification models from cytology-on-a-chip measurements that mirror diagnostic performance of gold standard approach involving tissue biopsy. MATERIALS AND METHODS: Measurements were recorded from 714 prospectively recruited patients with suspicious lesions across 6 diagnostic categories (each confirmed by tissue biopsy -histopathology) using a powerful new 'cytology-on-a-chip' approach capable of executing high content analysis at a single cell level. Over 200 cellular features related to biomarker expression, nuclear parameters and cellular morphology were recorded per cell. By cataloging an average of 2000 cells per patient, these efforts resulted in nearly 13 million indexed objects. RESULTS: Binary "low-risk"/"high-risk" models yielded AUC values of 0.88 and 0.84 for training and validation models, respectively, with an accompanying difference in sensitivity+specificity of 6.2%. In terms of accuracy, this model accurately predicted the correct diagnosis approximately 70% of the time, compared to the 69% initial agreement rate of the pool of expert pathologists. Key parameters identified in these models included cell circularity, Ki67 and EGFR expression, nuclear-cytoplasmic ratio, nuclear area, and cell area. CONCLUSIONS: This chip-based approach yields objective data that can be leveraged for diagnosis and management of patients with PMOL as well as uncovering new molecular-level insights behind cytological differences across the OED spectrum.
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Dispositivos Lab-On-A-Chip , Monitorização Fisiológica/métodos , Neoplasias Bucais/patologia , Automação , Biópsia/métodos , Feminino , Humanos , Masculino , Estudos ProspectivosRESUMO
UNLABELLED: HIV-1 is characterized by high genetic heterogeneity which is a challenge for developing therapeutics. Therefore, it is necessary to understand the extent of genetic variations that HIV is undergoing in North India. The objective of this study was to determine the role of genetic and functional role of Vif on APOBEC3G degradation. Vif is an accessory protein involved in counteracting APOBEC3/F proteins. Genetic analysis of Vif variants revealed that Vif C variants were closely related to South African Vif C whereas Vif B variants and Vif B/C showed distinct geographic locations. This is the first report to show the emergence of Vif B/C in our population. The functional domains, motifs and phosphorylation sites were well conserved. Vif C variants differed in APOBEC3G degradation from Vif B variants. Vif B/C revealed similar levels of APOBEC3G degradation to Vif C confirming the presence of genetic determinants in C-terminal region. High genetic diversity was observed in Vif variants which may cause the emergence of more complex and divergent strains. These results reveal the genetic determinants of Vif in mediating APOBEC3G degradation and highlight the genetic information for the development of anti-viral drugs against HIV. IMPORTANCE: Vif is an accessory HIV-1 protein which plays significant role in the degradation of human DNA-editing factor APOBEC3G, thereby impeding the antiretroviral activity of APOBEC3G. It is known that certain natural polymorphisms in Vif could degrade APOBEC3G relatively higher rate, suggesting its role in HIV-1 pathogenesis. This is the first report from North India showcasing genetic variations and novel polymorphisms in Vif gene. Subtype C is prevalent in India, but for the first time we observed putative B/C recombinants with a little high ability to degrade APOBEC3G indicating adaptation and evolving nature of virus in our population. Indian Vif C variants were able to degrade APOBEC3G well in comparison to Vif B variants. These genetic changes were most likely selected during adaptation of HIV to our population. These results elucidate that the genetic determinants of Vif and highlights the potential targets for therapeutics.
Assuntos
Citidina Desaminase/metabolismo , Recombinação Genética , Produtos do Gene vif do Vírus da Imunodeficiência Humana/fisiologia , Desaminase APOBEC-3G , Sequência de Aminoácidos , HIV-1/genética , Humanos , Índia , Dados de Sequência Molecular , Filogenia , Proteólise , Homologia de Sequência de Aminoácidos , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
6-Endo-dig-cyclization is an efficient method for the synthesis of 1,2-dihydroisoquinolines. We have synthesized few 1,2-dihydroisoquinolines having different functionality at the C-1, C-3, C-7, and N-2 positions for evaluation against HIV-1 integrase (HIV1-IN) inhibitory activity. A direct nitro-Mannich condensation of o-alkynylaldimines and dual activation of o-alkynyl aldehydes by inexpensive cobalt chloride yielded desired compounds. Out of 24 compounds, 4m and 6c came out as potent integrase inhibitors in in vitro strand transfer (ST) assay, with IC50 value of 0.7 and 0.8 µM, respectively. Molecular docking of these compounds in integrase revealed strong interaction between metal and ligands, which stabilizes the enzyme-inhibitor complex. The ten most active compounds were subjected to antiviral assay. Out of those, 6c reduced the level of p24 viral antigen by 91%, which is comparable to RAL in antiviral assay. Interestingly, these compounds showed similar ST inhibitory activity in G140S mutant, suggesting they can act against resistant strains.